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M. Levrero - Histone Acetyltransferase (HAT) autoregulatory loops in the regulation of DNA damage responses
modulation of chromatin-bound hSirT1 deacetylase
activity by the intracellular redox state with P1p73
promoter activity. Release of PCAF from hSirT1
repression favours the assembly of transcriptionally
active PCAF/E2F1 complexes onto the P1p73 promoter
and p53-independent apoptosis. These results
identify hSirT1 and PCAF as potential targets to
modulate tumor cell survival and chemoresistance
irrespective to p53 status.
Chromatin Immunoprecipitation-based
innovative reagents for a comprehensive
evaluation of the E2F1 transcriptome
Microarrays-based expression profiling studies have
led to the identification of hundreds potential direct
target genes activated and/or repressed by the different
members of the E2F family of transcription
factors. Genome-wide ChIP-chip experiments have
confirmed that several hundreds putative E2F binding
sites exist and are potentially bound throughout
the genome. We combined an extensive in silico
analysis with literature survey to select 327 putative
E2F1 direct target genes containing highly conserved
E2F/DP binding sites in their promoter or
first intron (i.e. -2500 to +1000 respect to tss). To
build up the E2F/DP dedicated microchip slide 3 different
45-50mer oligos for each of the 327 target
gene were designed. Each oligo was spotted in triplicate
and the resulting array printed 4 times on the
same slide for a total of 11772 spots. Gene ontology
analysis of the 327 selected genes is in agreement
with the notion that the spectrum of direct E2Fs target
genes might be far more variated than previously
thought: apoptosis (31), DNA damage (30), cell
cycle (71) cell growth (16), signal transduction (29);
transcription regulation (73), ubiquitination/Protein
degradation (7), protein/cell metabolism (30);
cytoskeleton/cell adhesion (38).
The E2F-ChIP array has been validated by
hybridization with anti-E2F1 and anti-Ac-H3
immunoprecipitated chromatin derived from untreat-
58
ed and doxorubicin treated U2OS cells. The analysis
of 2 independent ChIP-chip experiments has
revealed that E2F1 is present on 48% of the genes in
untreated cells. In cells exposed to apoptotic dosages
of doxorubicin, E2F1 is released from 33 out of 157
genes that were occupied in untreated genes (10% out
of 48%) whereas E2F is recruited onto 118 additional
target genes (36%) that were not bound in untreated
cells. There results indicate that DNA damage
results in a wide change in the spectrum of E2F1
bound promoters. E2F1 is bound before and after
treatment on 41% of the genes involved in apoptosis
and DNA repair included in the array and it is
recruited on additional 38% of these genes.
Interestingly, among the genes that regulate cell
cycle and whose expression is expected to be mostly
down regulated in cells undergoing DNA damageinduced
growth-arrest and apoptosis, only 17% loose
E2F1 after exposure to doxorubicin whereas E2F1
remains bound to 30% of these genes and is recruited
on 39% additional genes. The analysis of the correlation
between E2F1 promoter occupancy and gene
expression indicate that multiple mechanisms are
involved in the regulation of E2F target genes after
DNA damage including the recruitment of repressive
E2F1-containing complexes and the modulation
of E2F1-bound complexes by the recruitment of corepressors
and the release of coactivators. The E2F
ChIP-on-chip reagent we have developed and validated
and the knowledge that it generates will represent
a powerful tool to define the spectrum of genes targeted
by E2F1 to modulate cell death, senescence and
proliferation and to functionally characterize the
E2F transcriptome in vitro and in vivo.
Selected publications
Blandino G, Fanciulli M, Levrero M, Piaggio G.
The post-genomic era: workshop on chromatin
immunoprecipitation-related techniques. Cell Death
Differ. 2007, 14:1390-1.